Composition for preventing, ameliorating, and treating periodontitis

Abstract

To provide a component effective for preventing periodontitis, and improving and treating the symptoms, and a food product, a medicine and a quasi drug using the component.SOLUTION: A composition for oral cavity for preventing, improving and treating periodontitis which contains at least one or more of any intermediate metabolite in a metabolic pathway reaching flavonoid from phenylalanine via phenylpropanoid, or arctigenin as an effective component. It is desirable that the intermediate metabolite in the metabolic pathway reaching flavonoid from the phenylalanine via phenylpropanoid be a cinnamic acid derivative.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an ingredient effective for preventing, ameliorating symptoms of, and treating periodontitis, and to foods, pharmaceuticals, and quasi-drugs that use the ingredient. [Background technology]

[0002] According to the 2016 Dental Disease Survey (Ministry of Health, Labour and Welfare), over 30% of minors (aged 15-19) experience gingival bleeding, and over 30% of people aged 25-34 have periodontal pockets of 4mm or more, known as chronic periodontitis. Furthermore, the number of people with periodontal pockets of 4mm or more increases with age, reaching over 50% of people aged 55 or older. Chronic periodontitis can be said to be a national disease.

[0003] Chronic periodontitis is perceived as a cause of concern, such as swollen gums, periodontal bleeding, chewing pain, bad breath, receding gums, or tooth sensitivity. However, these are not merely mild subjective symptoms or simple inflammation; in severe cases, more than two-thirds of the alveolar bone is resorbed, causing tooth mobility and ultimately tooth loss, making it a major cause of a significant decline in quality of life in an aging society. When severe chronic periodontitis results in tooth mobility and loss, the only treatment available is periodontal surgery, and prevention and treatment before the condition worsens are essential to maintaining quality of life. However, periodontal treatment for mild chronic periodontitis is said to worsen symptoms (Non-Patent Document 1), and there is a need for methods of prevention and treatment that can prevent and treat the condition before it becomes severe or while it is still mild.

[0004] The most important point in preventing periodontitis or suppressing its worsening is plaque control, and removing plaque has been considered the basis for preventing and treating periodontal disease. Over the past 50 years, plaque control techniques have improved significantly, and numerous oral antibacterial agents have been developed, resulting in a dramatic reduction in the incidence of dental caries.

[0005] On the other hand, the incidence of chronic periodontitis, a representative periodontal disease, has not shown a decreasing trend in any region of the world when comparing 1990 and 2010 (Non-Patent Document 2). This means that chronic periodontitis cannot be prevented or treated at least by plaque control or control of oral bacterial flora, i.e., by sterilizing harmful bacteria, and there is a strong demand for fundamental methods of preventing and treating chronic periodontitis.

[0006] Although the mechanisms of onset and progression of chronic periodontitis have not been fully elucidated, the screening method for preventive and therapeutic ingredients to date has been based on their bactericidal activity against periodontal pathogens. Although numerous bactericides with clear bactericidal effects against periodontal pathogens have been developed to date, the incidence of chronic periodontitis has not decreased, suggesting that bactericidal activity is not directly related to the mechanism of onset of chronic periodontitis, and elucidation of the true mechanism of chronic periodontitis has been called for.

[0007] Recent years have seen significant progress in this research, revealing that the primary cause of chronic periodontitis is an increase in the proportion of fibroblasts present in gingival tissue that have a high rate of collagen degradation, resulting in the degradation of the connective tissue that makes up the gingiva. Furthermore, a three-dimensional culture method for periodontitis-associated fibroblasts (PAF) collected from the gingiva of patients with chronic periodontitis has been established, and technology has been established for screening compounds that inhibit the regression of collagen tissue (Non-Patent Documents 3-5, Patent Document 1). Using periodontitis-associated fibroblasts for screening, ongoing research is being conducted into preventive and therapeutic agents that address the mechanisms of chronic periodontitis.

[0008] The present inventors have already used a three-dimensional culture system of PAF to screen herbal medicines for those that inhibit collagen degradation. As a result, Scutellaria Root, Cinnamon Bark, and Poria Coccinella were found to be particularly effective. Furthermore, the effectiveness of licorice, Chinese Root, Cnidium Rhizome, Boufu Root, Scutellaria Baicalensis, Coptis Rhizome, Onji, Kyokatsu Root, Magnolia Root, Euonymus Root, Ginger Root, Calamus Root, Senkotsu Root, and Xinji was also found, raising hopes for the development of new preventive and therapeutic drugs containing active ingredients from herbal medicines (Patent Document 2). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Patent Application Laid-Open No. 2010-220561 [Patent Document 2] Japanese Patent Application Laid-Open No. 2011-256136 [Patent Document 3] Japanese Patent Application Laid-Open No. 2011-162455 [Patent Document 4] Japanese Patent Application Laid-Open No. 2013-216589 [Non-patent literature]

[0010] [Non-Patent Document 1] Badersten, A. et al., 1985, J. Clinical Periodontology, Vol.12, pp.351-359. [Non-patent document 2] Kassebaum, NJ et al., 2014, J. Dent. Res. Vol.93(11), pp.1045-1053. [Non-patent document 3] Ohshima, M. et al., 2010, J. Dent. Res., Vol. 89(11), pp.1315-1321. [Non-patent document 4] Ohshima, M. et al., 2016, J. Clin. Periodontol., Vol.43, pp.128-137. [Non-Patent Document 5] Horie, M. et al., 2016, Scientific Reports, 6:33666, DOI:10.1038 / srep33666. [Non-patent document 6] Ryoko Saijo, 2012, Tea Industry Research Report, No. 114, p.79-88. [Non-Patent Document 7] Ohshima et al., 1994, J. Periodontal Res., Vol.29, pp.421-429. Summary of the Invention [Problem to be solved by the invention]

[0011] Regarding the prevention and treatment of chronic periodontitis, plaque control and methods of eliminating oral bacteria (especially so-called bad bacteria) have generally been proposed. Furthermore, at the end of fiscal year 2019, a functional food product that improves oral flora was registered with the Consumer Affairs Agency. However, while these methods are successful prevention and treatment strategies for dental caries, they do not address the mechanism of chronic periodontitis onset described above. The objective of the present invention is to provide a composition for preventing, symptomatic improvement, and treatment of gingival recession caused by the degradation of collagen fibers by gingival fibroblasts, the main cause of chronic periodontitis.

[0012] Patent Document 2 discloses many herbal medicines as preventive and therapeutic agents that address the mechanism of chronic periodontitis onset. These herbal medicines contain a variety of components, and are thought to have multiple active ingredients. If the active ingredients contained in these herbal medicines could be identified, not only would more effective preventive and therapeutic agents be obtained, but the mechanism of action behind periodontitis could also be elucidated. The objective of the present invention is to identify active ingredients contained in herbal medicines that are effective in preventing, alleviating symptoms, and treating chronic periodontitis, and to provide even more effective foods and pharmaceuticals. [Means for solving the problem]

[0013] The present invention relates to an oral composition for preventing, ameliorating, and treating periodontitis, which contains the following active ingredients: (1) An oral composition for preventing, improving, and treating periodontitis, which contains at least one of the intermediate metabolites in the metabolic pathway from phenylalanine to flavonoids via phenylpropanoids, or arctigenin as an active ingredient. (2) The oral composition according to (1), wherein the intermediate metabolite in the metabolic pathway from phenylalanine to flavonoid via phenylpropanoid is a cinnamic acid derivative. (3) The oral composition according to (2), wherein the cinnamic acid derivative is hydroxycinnamic acid. (4) The oral composition according to (3), wherein the hydroxycinnamic acid is coumaric acid, coumarin, chlorogenic acid, caffeic acid, or ferulic acid. (5) The oral composition according to (1), wherein the flavonoid is apigenin or cyanidin. (6) A health food, a medicine, a quasi-drug, or a cosmetic comprising the oral composition according to any one of (1) to (5). (7) The health functional food, medicine, quasi-drug, or cosmetic according to (6), wherein the health functional food is provided in the form of a supplement or food or drink, the medicine or quasi-drug is provided in the form of a chewable tablet, lozenge, mouthwash, ointment, film, ready-to-use preparation, mouthwash, or toothpaste, and the cosmetic is provided in the form of toothpaste. [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows the results of analyzing the effect of cinnamic acid derivatives on the collagen degradation inhibitory ability using a collagen gel three-dimensional culture system. [Figure 2] Microscope images showing the state of cells in collagen gels with the addition of green coffee, chlorogenic acid, and p-coumaric acid. DMSO was added as a control. Microscope images of each gel stained with HE and Sirius Red are shown. [Figure 3] Microscope images showing the state of cells in collagen gels with the addition of caffeic acid and ferulic acid. DMSO was added as a control. Microscope images of each gel stained with HE staining, Sirius Red staining, and anti-vimentin antibody are shown. [Figure 4] The results of analyzing the effects of arctigenin, apigenin, and cyanidin on the collagen degradation inhibitory ability using a collagen gel three-dimensional culture system are shown. [Figure 5] Microscopic images showing the state of cells in collagen gels with the addition of arctigenin, apigenin, and cyanidin. DMSO was added as a control. Microscopic images of each gel stained with HE and Sirius Red are shown. [Figure 6] Diagram showing the metabolic pathway from phenylalanine to flavonoids via phenylpropanoids. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present inventors analyzed the components of herbal medicines that inhibit collagen degradation and predicted candidate compounds that may have the ability to inhibit collagen degradation. Among the herbal medicines that inhibit collagen degradation, herbal medicines containing cinnamon acid derivatives, such as cinnamon bark, Cnidium officinalis, Boufu, Coptis chinensis, Onji, Kyokatsu, and Xinji, were found. Therefore, we first investigated the possibility that cinnamon acid derivatives have the ability to inhibit collagen degradation.

[0016] The results, detailed below, revealed that cinnamic acid derivatives have the ability to inhibit collagen degradation. Among the cinnamic acid derivatives, hydroxycinnamic acids, specifically p-coumaric acid, chlorogenic acid, caffeic acid, and ferulic acid, were particularly effective in inhibiting collagen degradation. Furthermore, although the results for coumarin are not shown here, coumarin has also been confirmed to inhibit collagen gel degradation. Coumarin is a compound produced by the cyclization reaction of coumaric acid and is easily converted from coumaric acid. While coumarin itself has the ability to inhibit collagen degradation, given that coumaric acid has a strong inhibitory effect on collagen gel degradation and that coumaric acid is produced from coumaric acid in vivo, it is likely that if coumarin is present in the raw materials, it will be converted to coumaric acid and thus have the ability to inhibit collagen degradation.

[0017] Although the mechanism by which cinnamic acid derivatives inhibit collagen degradation is unclear, the effectiveness of several cinnamic acid derivatives suggests that intermediate metabolites in the metabolic pathway from phenylalanine to flavonoids via phenylpropanoids may be effective (Non-Patent Document 6). Similar analysis was then performed on apigenin, an anthocyanin derivative, which is one of the intermediate metabolites in this metabolic pathway other than cinnamic acid derivatives, and revealed that it has the ability to inhibit collagen degradation. In addition to the cinnamic acid derivatives that were actually analyzed, the pathway synthesizing flavonoids from phenylalanine via phenylpropanoids contains various compounds, including naringenin, luteolin, kaempferol, quercetin, pelargonidin, cyanidin, and delphinidin (see Figure 6). Further analysis is needed to determine whether these compounds have the ability to inhibit collagen degradation, but given their structural similarities, they are likely to be effective.

[0018] Furthermore, the inventors discovered that arctigenin also has the effect of inhibiting collagen degradation. Arctigenin is a lignan and is not included in the metabolic pathway described above. However, its synthetic pathway, the cinnamic acid monolignol pathway, starts from phenylalanine and synthesizes coniferyl alcohol, the starting point for lignan synthesis, via cinnamic acid, caffeic acid, and ferulic acid. Since part of the synthetic pathway shares many intermediate metabolites that have been shown to have an inhibitory effect on collagen degradation, analysis of arctigenin revealed its ability to inhibit collagen degradation. Regarding lignans, analysis has been limited to arctigenin, but it is possible that lignans derived from coniferyl alcohol may have similar activity.

[0019] These compounds can be used alone as active ingredients in health foods or pharmaceutical compositions. Intermediate metabolites in the metabolic pathway from phenylalanine to flavonoids via phenylpropanoids, particularly cinnamic acid derivatives or flavonoids with proven efficacy, such as apigenin and cyanidin, can also be used as active ingredients. These compounds can be used alone or in combination, or glycosides can be used. Examples of commercially available extracts containing cinnamic acid derivatives include "Peanut Seed Coat Extract" (trade name) manufactured by Access One Co., Ltd., "Garlic Extract" (Bioactives Japan Co., Ltd.), "Green Coffee Bean Extract-P" (trade name) manufactured by Oryza Oil & Fat Chemical Co., Ltd., "Coffee Bean Extract (Chlorogenic Acid)" (trade name) manufactured by Bioactives Japan Co., Ltd., "Ferulic Acid" (trade name) manufactured by Oryza Oil & Fat Chemical Co., Ltd., and "BioBenefiti F (Artichoke Leaf Extract)" (trade name) manufactured by Ichimaru Pharcos Co., Ltd.

[0020] Alternatively, extracts of plants containing large amounts of these compounds may be used. For example, p-coumaric acid is known to be found in peanuts, tomatoes, carrots, garlic, lemons, olives, and bamboo shoots; coumarin is found in cherry leaves, cinnamon, tonkama beans, angelica, angelica tree, and oranges; chlorogenic acid is found in coffee beans, mugwort, eggplant, cranberries, edible chrysanthemums, caraway seeds, and sweet potato stems and leaves; caffeic acid is found in coffee beans, olives, grape leaves, sweet potato stems and leaves, sweet potato shochu lees, spinach, artichokes, date seeds, perilla extract, sweet tea extract, ginkgo extract, eggplant, and burdock; and ferulic acid is found in rice, wheat, rye, barley, soybeans, adzuki beans, corn, sesame, coffee beans, apples, artichokes, peanuts, oranges, pineapples, green onions, evening primrose, jojoba, wine, spinach, bamboo shoots, and date seeds. It is known that apigenin is contained in large amounts in guava, garlic, celery, parsley, and vervain, and that arctigenin is contained in large amounts in burdock, particularly in its seeds, sprouts, and plants of the genus Forsythia, which are used as tea leaves. Therefore, extracts of these plants or their processed products may be used.

[0021] Apigenin is an anthocyanin derivative, and examples of commercially available extracts containing anthocyanin derivatives include "Blackberry Extract Powder" (trade name) manufactured by Koshin Bussan Co., Ltd., "Black Rice Extract-P" (trade name) and "Maqui Berry Extract-P35" (trade name) manufactured by Oryza Oil & Fat Chemical Co., Ltd., "Aronia Extract Powder" (trade name) manufactured by BGG Japan Inc., "Elderberry Extract 30.5%" (trade name) manufactured by Kaiyu Shoji Co., Ltd., "Bilberry Extract Powder (25% product)" (trade name) manufactured by Newtonex Co., Ltd., and "Cassis Extract Powder" (trade name) manufactured by Matsuura Pharmaceutical Co., Ltd.

[0022] To achieve even greater efficacy, the oral composition of the present invention may be combined with a cinnamic acid derivative-containing extract and an anthocyanin-containing extract. Other active ingredients, such as phosphodiesterase inhibitors (Patent Document 3) and tyrosine kinase inhibitors (Patent Document 4), previously discovered by the inventors through screening for their ability to inhibit collagen degradation, may also be added. Active ingredients reported to be effective against chronic periodontitis may also be added. Examples of such active ingredients include probiotics such as lactic acid bacteria, CoQ10, astaxanthin, isoflavones, protamine degradation products, sugar alcohols, tea polyphenols found in oolong tea, licorice polyphenols, trace volatile components of wasabi, eucalyptus extract, bay leaf extract, polyglutamic acid, and kumazasa leaf extract. Combining multiple active ingredients can be expected to produce additive or synergistic effects.

[0023] Compounds with collagen degradation inhibitory activity can be used in specific categories such as health functional foods, health foods (supplements), quasi-drugs, pharmaceuticals, and cosmetics. In particular, they are preferably used in the form of oral care products that can be used continuously from the pre-disease or mild periodontitis stage, or in the form of foods and beverages (drinks and foods) such as health functional foods, functional foods, and health foods. Health functional foods refer to foods for specified health uses and foods with nutrient functions under Japan's system, while functional foods refer to foods whose functions are indicated by the business operator themselves based on scientific evidence. There are also so-called health foods and supplements that do not fall into either category. Regardless of the category, it is desirable for the compounds to be in a form that allows for continuous use. They can also be used as pharmaceuticals, quasi-drugs, and as ingredients in cosmetics such as toothpaste.

[0024] When the oral composition of the present invention is used as a periodontal supplement or health food, the target pre-disease or pre-lesion is not particularly limited, as long as periodontal collagen degradation is considered to be one of the causes. The target gingiva is preferably in a state where there is no gingival redness or swelling, the periodontal pocket depth is 3 mm or less, there is no bleeding on probing (BOP), and there is no obvious bad breath. In such pre-disease or pre-symptomatic stages, the onset of periodontitis can be suppressed. In pre-lesion or mild periodontitis, the progression of periodontitis can be delayed, and the progression to symptoms that significantly reduce quality of life, such as tooth mobility and tooth loss, can be suppressed.

[0025] Supplements are a type of health food, containing concentrated specific ingredients and in a form that is not easily confused with ordinary foods. When the oral composition of the present invention is in the form of a supplement, it may be in the form of, but is not limited to, granules, tablets, soft capsules, chewable tablets, films, lozenges, liquids, mouthwashes, candies, gums, or gummies. The supplement of the present invention may further contain pharmaceutically acceptable carriers or additives such as preservatives, excipients, buffers, surfactants, binders, disintegrants, lubricants, colorants, flavorings, solubilizers, suspending agents, and coating agents. When used as a periodontal supplement, it is desirable to take it in a clean oral cavity, and it is particularly preferable to take it after rinsing the oral cavity with toothpaste or mouthwash.

[0026] When the embodiment is a health functional food, the food may contain, as an active ingredient, a cinnamic acid derivative, or an extract, concentrated extract, or dried extract (spray-dried, freeze-dried, etc.) obtained from a plant containing a large amount of cinnamic acid derivative. Specific forms of health functional foods are preferably foods that remain in the mouth for a long time and can be stored for a long time, such as gum, candy, or gummy candy. Furthermore, liquid foods that can be consumed frequently, such as tea drinks, coffee drinks, and milk drinks, are also preferred, but are not limited to these.

[0027] When the composition of this embodiment is used as a pharmaceutical or quasi-drug for treatment, the target disease is not particularly limited as long as periodontal collagen degradation is considered to be one of the causes. However, chronic periodontitis, particularly chronic periodontitis that becomes chronic and causes severe gingival recession, is preferred. Furthermore, administration methods include topical application, injection, oral administration, and injection into periodontal pockets. In particular, the pharmaceutical of the present invention is preferably for oral or buccal administration. The pharmaceutical of the present invention may be in the form of, but is not limited to, tablets, capsules, chewable tablets, lozenges, gels, films, ready-to-use preparations, granules, fine granules, powders, sustained-release preparations, suspensions, emulsions, syrups, elixirs, liquids, ointments, patches, mouthwashes, sprays, and the like. In addition, the pharmaceutical product according to the present invention may further contain a pharmaceutically acceptable carrier or additive such as a preservative, excipient, buffer, surfactant, binder, disintegrant, lubricant, colorant, flavoring agent, solubilizing agent, suspending agent, or coating agent.

[0028] Furthermore, specific product forms of compositions used as quasi-drugs or cosmetics for the prevention or treatment of mild periodontitis may be in the form of products used for oral care, such as dentifrices, liquid toothpaste, gum massage gels, mouthwashes, etc. It goes without saying that even in the case of product forms of compositions for the prevention or treatment of mild periodontitis classified as quasi-drugs or cosmetics, carriers or additives may be included.

[0029] The oral compositions of the present invention contain cinnamic acid derivatives, such as chlorogenic acid, p-coumaric acid, caffeic acid, ferulic acid, or their pharmaceutically acceptable salts or glycosides, as well as flavonoids such as apigenin, cyanidin, or arctigenin, in amounts ranging from 1 to 2,000 mg / day, preferably 5 to 1,000 mg / day, and more preferably 10 to 200 mg / day. Alternatively, plant extracts containing high amounts of these components, or components derived therefrom, such as coffee bean extract, may be used in amounts similar to those listed above. These components may be contained alone or in combination, although this is not a limitation.

[0030] Furthermore, the compositions of the present invention for preventing, ameliorating, and treating periodontitis target pre-disease and mild periodontitis. Here, chronic periodontitis is defined as a periodontal pocket depth (PPD) of more than 4 mm or stage I or higher according to the 2018 New Classification of Periodontitis by the American Academy of Periodontology and the European Federation of Periodontology. However, there is a gray area in the definition of the pre-disease state of chronic periodontitis, and the boundary between pre-disease and disease is not always clear. In this application, pre-disease is generally considered to be a state in which periodontal pocket depth is less than 3 mm and no periodontitis treatment is performed, or a state milder than stage I and with a clinical attachment level (CAL) of less than 1 mm. In other words, the pre-disease state of chronic periodontitis refers to a reversible condition that can be restored to a healthy state with some kind of treatment.

[0031] That is, at the stage of gingivitis, the periodontal pocket is a pseudopocket without attachment loss, and the clinical pocket depth (PPD) is 1 to 3 mm. Also, at the stage of gingivitis, mild inflammation of the gums and bleeding on probing (BPD) from the periodontal pocket are observed. Among these, the present invention is effective against periodontitis that progresses from gingivitis, and may inhibit the progression of gingivitis to chronic periodontitis, bringing the gums closer to a healthy state.

[0032] In the mild periodontitis stage, attachment loss occurs and the periodontal pocket becomes a true pocket, with clinical pocket depth exceeding 3 mm. Furthermore, in the periodontitis stage, gingival inflammation and BOP are observed, and bone resorption begins. If the progression of this periodontitis is not suppressed, the symptoms will progress further, leading to tooth mobility and loss, resulting in a significant decline in quality of life. Although the present invention cannot restore attachment loss that occurs in mild periodontitis, by suppressing collagen degradation, it can suppress or slow the progression of the periodontal condition and prevent a decline in quality of life.

[0033] In addition, in this application, prevention is defined as maintaining a pre-disease state or bringing the state closer to a healthy state so that a definitive diagnosis of chronic periodontitis is not made, for example, a state in which the periodontal pocket is less than 3 mm but there is concern that it is gradually deepening, or a state that cannot be said to be chronic periodontitis but which may lead to a definitive diagnosis of chronic periodontitis if left untreated, is maintained in a state in which a definitive diagnosis is not made, or as bringing the state closer to a healthy state.

[0034] [Example] Study using a collagen gel three-dimensional culture system The present invention will be explained below while showing the results. 1. Cell Preparation Analysis was performed using fibroblasts and epithelial cells obtained from periodontal tissue using collagen gel three-dimensional culture technology. Gingival fragments that were removed during surgical procedures for the treatment of periodontitis or periodontal surgery and no longer needed were separated into connective tissue and epithelial tissue and then finely chopped. The epithelial tissue fragments were treated with dispase and then placed on a plate, and gingival epithelial cells exogenous from the fragments were subcultured as the first generation (Non-Patent Document 7). Similarly, gingival connective tissue was shredded and placed on a plate, and fibroblasts exogenous from each fragment were subcultured as the first generation. Multiple cell populations were obtained from the same specimen. Collagen gels were constructed using the subcultured gingival epithelial cells and gingival fibroblasts and used for analysis. Cell populations obtained from the connective tissue were screened using collagen gel three-dimensional culture to identify fibroblasts with high collagen degradation ability.

[0035] 2. Collagen Gel Construction A collagen mixture solution was prepared by mixing Cell Matrix Type-A (Nitta Gelatin), 5x DMEM, reconstitution buffer (Nitta Gelatin), and a test substance such as a cinnamic acid derivative, or the solvent DMSO as a control. Periodontitis-derived fibroblasts were suspended in this collagen mixture, and the sol was allowed to gel for 30 minutes in a 6-well plate to form a collagen gel containing periodontitis-derived fibroblasts. Next, the epithelial cells prepared above were dispersed with trypsin and seeded on the collagen gel to form an epithelial cell layer, creating a three-dimensional collagen gel.

[0036] 3. Observation of collagen gel contraction Collagen gels containing epithelial cells and periodontitis-derived fibroblasts were floated from the bottom of the plate 24 hours after collagen gel formation (culture day 1), and suspension culture was initiated. At the start of suspension culture, the test substance or solvent was added again to the culture medium to adjust the test substance concentration to the appropriate level based on the volume of epithelial cells seeded the previous day. Suspension culture was continued for approximately 5 to 7 days, and the contraction level of the collagen gel was observed to analyze whether the test substance inhibited collagen degradation.

[0037] Prior to the 3D culture analysis using collagen gel, the cytotoxicity of the added compounds was examined using 2D cultured fibroblasts. The compounds analyzed included cinnamic acid derivatives found in herbal medicines, as well as ferulic acid, a cinnamic acid derivative with a similar structure, p-coumaric acid, chlorogenic acid, and caffeic acid. Furthermore, we analyzed a green coffee bean extract (Green Coffee Bean Extract-P (trade name, Oryza Oil & Fat Chemical Co., Ltd.)), which is rich in caffeic acid. Green coffee bean extract is a powder obtained by extracting coffee beans, i.e., the seeds of the coffee tree (Coffea canephora) in the Rubiaceae family, with aqueous ethanol. Quantitative analysis of this product revealed that it is a water-soluble extract containing more than 24.0% chlorogenic acid and more than 45.0% chlorogenic acids.

[0038] All compounds were dissolved in DMSO and added to the culture medium at a maximum volume of 1 / 1000. No cytotoxicity was observed up to 200 μg / ml of green coffee bean extract, 20 μM of caffeic acid, 100 μM of ferulic acid, 20 μg / ml of chlorogenic acid, and 300 μg / ml of p-coumaric acid.

[0039] Therefore, we observed the level of collagen gel contraction using three-dimensional collagen gels containing each compound at a non-cytotoxic concentration range to see whether they inhibited PAF's collagen degradation ability. Each compound and control pair shows results using cells derived from the same patient. All compounds were shown to inhibit PAF's collagen gel degradation (Figure 1). All cinnamic acid derivatives, as well as green coffee bean extract containing cinnamic acid derivatives, were also shown to inhibit collagen gel degradation. In particular, ferulic acid and p-coumaric acid were found to have a significant inhibitory effect.

[0040] After the collagen gel 3D culture analysis, the gels were fixed in formalin using standard methods and thinly sliced. The groups containing green coffee bean extract, chlorogenic acid, and p-coumaric acid were then stained with HE and Sirius Red (Figure 2), while the groups containing caffeic acid and ferulic acid were stained with HE, Sirius Red, and anti-vimentin antibody (Figure 3). The HE staining results showed that cells within the collagen gel were visible, while Sirius Red staining revealed the orientation of collagen fibers, and immunostaining with anti-vimentin antibody revealed the cytoskeleton of fibroblasts, which is composed of vimentin.

[0041] When green coffee bean extract or chlorogenic acid was added, exposure to air during fixation tended to shift epithelial cells toward the center, resulting in a thin collagen gel spreading around the periphery. While both green coffee bean extract and chlorogenic acid inhibited collagen gel degradation during culture, air exposure during fixation likely accelerated collagen degradation. Microscopic images of gels containing chlorogenic acid or green coffee bean extract revealed voids. However, while the control exhibited noticeable voids in both HE and Sirius Red staining, green coffee bean extract significantly reduced voids compared to the control, demonstrating its inhibition of collagen degradation. Furthermore, gels containing chlorogenic acid clearly inhibited collagen degradation in suspension cultures, but although voids were visible in microscopic images, they were fewer in number than the control (Figure 2). Because the gel morphology changed upon exposure to air when chlorogenic acid or green coffee bean extract was added, this change is likely due to a loss of antioxidant activity due to air exposure. When chlorogenic acid or green coffee bean extract is used, it is believed that collagen degradation can be further suppressed by adding an antioxidant or the like.

[0042] On the other hand, the gel containing p-coumaric acid showed no damage to cells and a significant ability to inhibit collagen degradation, as shown in Figure 1. Furthermore, as shown in Figure 2, HE staining showed no voids within the gel, and Sirius Red staining also showed a clear orientation of collagen fibers.

[0043] As shown in Figure 1, caffeic acid was found to weakly inhibit collagen degradation in an analysis using collagen gel-gel 3D culture. HE staining, Sirius Red staining, and immunostaining with anti-vimentin antibody supported this, demonstrating suppression of voids, collagen degradation, and maintenance of the cytoskeleton compared to the control (Figure 3). Ferulic acid significantly inhibited collagen degradation (Figure 1), but collagen fibers and the cytoskeleton, including vimentin, were also maintained compared to the control (Figure 3).

[0044] These results demonstrated that cinnamic acid derivatives, especially coumaric acid and ferulic acid, have a strong inhibitory effect on collagen degradation. Since several cinnamic acid derivatives exhibit collagen degradation inhibitory effects, we suspected that intermediate metabolites in the metabolic pathway from phenylalanine to flavonoids via phenylpropanoids might be effective (see Figure 6). Apigenin, another intermediate metabolite in this metabolic pathway other than cinnamic acid derivatives, was analyzed in a similar manner. As shown below, apigenin exhibited a strong inhibitory effect on collagen degradation, so we also analyzed cyanidin, an anthocyanin derivative with a similar structure. Furthermore, arctigenin was also analyzed because a portion of the pathway synthesizing coniferyl alcohol, the starting point for lignan synthesis, via phenylalanine, cinnamic acid, caffeic acid, and ferulic acid shares many of the intermediate metabolites with the inhibitory effect on collagen degradation.

[0045] We investigated the collagen gel degradation ability of arctigenin, apigenin, and cyanidin. First, we examined the cytotoxicity of fibroblasts cultured in 2D with arctigenin, apigenin, and cyanidin. Subsequently, we performed 3D collagen gel cultures without cytotoxicity and analyzed the results. We added each compound to 3D collagen gels at concentrations of 100 μg / ml for arctigenin, 10 μg / ml for apigenin, and 20 μg / ml for cyanidin. We then observed the contraction level of the collagen gels and observed whether the compounds inhibited PAF's collagen degradation. Arctigenin and apigenin were dissolved in DMSO, and cyanidin was dissolved in ethanol. Similar to DMSO, ethanol was added to the medium at a maximum volume of 1 / 1000. The control gels were DMSO-added. The results are shown in Figure 4.

[0046] The top row of Figure 4 shows the state of the cells after formalin fixation, and the bottom row shows the state of the cells before fixation. Arctigenin and apigenin were found to have a significant collagen degradation inhibitory effect. Cyanidin caused a slightly weaker degree of contraction than the control, suggesting that it may have a weak collagen degradation inhibitory effect.

[0047] Furthermore, after the collagen gel 3D culture analysis, the gels were fixed in formalin using standard methods, sliced, and then stained with HE and Sirius Red (Figure 5). The results of Sirius Red staining showed that the collagen fibers were neatly oriented in the gels treated with arctigenin or apigenin, demonstrating that these compounds have the effect of inhibiting collagen degradation. Furthermore, a decrease in vacuoles around the cells was observed in the gels to which cyanidin was added, suggesting that they have a weak inhibitory effect on collagen degradation.

[0048] Figure 6 shows the metabolic pathway from phenylalanine to flavonoids via phenylpropanoids. The structural formulas of the starting phenylalanine and the analyzed compounds are shown. Collagen degradation inhibitory effects were observed in the cinnamic acid derivatives p-coumaric acid, caffeic acid, chlorogenic acid, and ferulic acid, as well as the flavonoids apigenin and cyanidin, which are synthesized from p-coumaric acid via naringenin. If metabolic pathways are involved, other compounds shown in Figure 6 that could not be analyzed here, such as naringenin, luteolin, kaempferol, quercetin, pelargonidin, and delphinidin, are likely to also have collagen degradation inhibitory effects. Furthermore, since arctigenin also exhibited collagen degradation inhibitory effects, it is highly likely that intermediate metabolites in the pathway from ferulic acid to coniferyl alcohol via lignan synthesis are also active.

[0049] Because the analysis system using collagen gel three-dimensional culture is a living model of periodontitis onset, it is believed that periodontitis can be treated by applying compounds present in the metabolic pathway from phenylalanine, such as cinnamic acid derivatives, to flavonoids via phenylpropanoids, or arctigenin to the affected area of ​​periodontitis. Oral compositions containing cinnamic acid derivatives, flavonoids, and arctigenin that are in line with the mechanism of periodontitis onset are believed to be particularly effective in treating mild periodontitis in which collagen degradation in the tissue has not progressed significantly, and in preventing periodontitis.

Claims

1. An oral composition for inhibiting collagen tissue regression caused by periodontitis, comprising coumaric acid as an active ingredient.

2. The oral composition for inhibiting collagen tissue regression according to claim 1, further comprising arctigenin.

3. An oral composition for inhibiting collagen tissue regression as described in claim 1 or 2, which is a health functional food, a medicine, a quasi-drug, or a cosmetic.

4. The health functional food is a supplement or a food or drink, The pharmaceutical or quasi-drug is a chewable agent, a lozenge, a mouthwash, an ointment, a film, a ready-to-use agent, a mouthwash, or a toothpaste, 4. The oral composition for inhibiting collagen tissue regression according to claim 3, wherein the cosmetic is a dentifrice.

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